血小板低温保存的初步研究

血小板输注应用的进展,要求体外长期保存血小板,低温保存是目前公认细胞长期保存的最好方法。本研究用体外回收率、血小板聚集试验、血小板低渗休克试验与透射电镜观察等手段,研究了不同降温方法与冻存条件下血小板的活力。结果发现:(1)用自动控温装置降温效果最好。(2)选用5％DMSO低温保护剂与1℃/min降温率为最好冻存条件,此时70％以上血小板活力得以保存。(3)血小板低温损伤主要发生在降温时融合热释放期(-6℃以上)。(4)-20℃冻存是不可取的。(5)血小板聚集功能在冻存后严重下降,有必要进一步研究解决方法。     

冰冻保存血小板再浓缩及临床应用

国内外就血小板冰冻保存的方法已进行了较多的研究 ,目前较典型的方法有 3类 :一是在富血小板血浆中加入终浓度为 4%的二甲基亚砜 ( DM-SO) ,- 80℃冻存 [1,2 ] 。二是先制备成浓缩血小板 ,再加入终浓度 4%～ 6%的 DMSO,- 80℃冻存 [3 ,4] ;或在浓缩血小板中加入 1 2 %的高浓度保护剂冻存 ,临用时需洗涤血小板[5] 。三是近年来开始应用的冰冻保存机采血小板。保存富血小板血浆 ,由于血浆量大 ,DMSO含量较多 ,对人体有一定副作用 ,限制了临床输注剂量 ,影响临床疗效。而先制成浓缩血小板后加 DMSO冻存的方法 ,由于高浓度的 DMSO在加…     

含6%二甲基亚砜的血小板的冷冻及-80℃或-135℃保存无需解冻后处理

在0.9％氯化钠(NaCl)中以27％二甲基亚砜(DMSO)处理人类单个献血员和狒狒血浆中的血小板,终浓度为6％,并浓缩以除去含95％DMSO的上清。将血小板重悬浮于300ml聚氯乙烯(PVC)袋中余下的10ml溶液或500ml乙烯乙基乙酸(EVA)袋中余下的15ml溶液中,外包有聚酯袋,置于一纸盒,并以2～3℃/min的速度在机械式冻箱中冷至～80℃。PVC袋冻存于-80℃而EVA袋冻存于-135℃冷冻箱中。每单位血小板约含600mgDMSO,将其解冻,并以10ml 0.9％NaCl稀释,然后输注。F—T回收率为85％,流动血细胞计数仪测得70％的为完整血小板而30％的为血小板微聚集。两只狒狒和一名正常志愿献血者     

二甲基亚砜血小板冻存剂的制备及质量控制

目的探讨二甲基亚砜(DMSO)血小板冻存剂优良的制备及质量控制方法。方法将分析纯DMSO进行除菌过滤后,样品分别以126℃30min一次、126℃30min二次湿热灭菌和180℃3h干热灭菌3种方法进行对比试验,并建立产品的性状、鉴别、无菌、热原、急性毒性、溶血试验、相对密度、折光率8个项目的检和质控方法。结果126℃30min二次湿热灭菌法制备的制剂各项检查均合格。结论本法制备DMSO血小板冻存剂质量稳定,质控方法可靠。     

单采血小板冷冻保存的质量评价

[目的]研究单采血小板冰冻保存。[方法]在单采血小板中加入二甲基亚砜(DMSO)，终浓度为5％，-120℃保存。观察冰冻一周，一个月后血小板聚集功能和回收率的改变、血小板低渗休克反应回复率的变化。[结果]冻存一周，一个月后单采血小板仍具有较高的回收率，血小板聚集功能和低渗休克反应回复率有一定下降。[结论]单采血小板冰冻保存是可行的，具有较好的疗效。贮存期可以延长。     

加入第二信使效应剂,降低二甲亚砜浓度的单一供者血小板的低温保存提高血小板体外机能活性

背景 细菌污染的可能性限制了血小板在22℃5天后的保存,这问题能通过低温贮存而允许血小板长期保存来解决。已经证实了含第二信使效应剂的混合液(血小板保存液)的血小板,冷藏保存后血小板保持良好的体外功能活性。分析第二信使效应混合液在冷藏期间保护血小板和减少低温保护剂的需要量应进行认真分析。方法 将新鲜单一供血者血小板单位(n=8)分成3个样本,并分别加入6％的二甲亚砜(DMSO),2％的DMSO,或血小板保存液和2％DMSO,将样本直接放入-80℃的冷藏箱中保存一个星期,待融化后分析他们的体外功能活性。结果用血小板贮存液和2％DMSO低温保存的血小板在统计学上显示在保持功能活性和存活力——包括细胞数,盘状细胞     

冰冻血小板制备研究

[目的]探讨冰冻单采血小板制备的可行性,为其临床应用提供可靠依据.[方法]通过认真选择献血员,单采血小板,并对40袋新鲜血小板进行计数等质控,用二甲基亚砜（DMSO）作冰冻保护剂,-80℃深低温保存1年内,解冻后观察外观,分别进行计数、计算回收率、测定PH值、粘附率、无菌试验,并且与新鲜血小板进行比较.[结果]-80℃保存冰冻单采血小板1年内血小板计数、pH值、粘附率与冻前新鲜血小板进行比较差异无显著性（P〉0.05）;血小板平均体积、血小板体积分布宽度冰冻前后差异有显著性（P〈0.01）,保存1年内平均回收率为90.49% , 无菌实验无细菌生长.[结论]-80℃深低温保存冰冻血小板质量达到标准要求,可以应用于临床.     

血小板保存新策略

血小板在常温(22℃±2℃)连续振荡下保存,对细菌生长极有利,从而易使血小板遭受细菌污染,并且因为保存期短,不能适应血小板临床需求量的迅速增加[1].解决这一问题的最好办法就是降低血小板的储存温度,进行血小板冷藏保存 [2].血小板冰冻保存可使血小板的保存期延长,但冻存后的血小板存活率明显降低且冰冻保护剂二甲基亚砜(DMSO)对受者具有毒副作用,在洗涤去除DMSO的过程中,血小板易激活、损伤[3].     

-80℃保存机采浓缩血小板的研究

目的:探讨–80℃冰冻保存机采浓缩血小板的功能及止血效果。方法:随机抽取冰冻保存3个月之内、3～6个月之间、6～9个月之间的冰冻血小板样品于42℃解冻后进行相关实验,比较不同保存时期冰冻血小板的功能及回收率;分血液病组和非血液病组观察患者输注冰冻血小板24h后出血症状的控制及校正血小板增高指数CCI值。结果:冻存3个月内、3～6个月的两组冰冻血小板的黏附功能、聚集功能与新鲜机采血小板相比差异均无显著性(P>0.05),而冻存6～9个月的冰冻血小板的黏附功能与新鲜机采血小板相比则差异有高度显著性(P<0.01)。3组冰冻血小板的第Ⅲ因子活性测定与新鲜血小板相比,均在正常范围,平均回收率为85.2%。两组患者在输注冰冻血小板24h后能有效控制出血症状的比率平均为93.3%;而CCI值≥10000/μL的比率,非血液病组显著高于血液病组。结论:5%二甲基亚砜-80℃冰冻保存半年以内的机采浓缩血小板具有良好的止血功能。     

以尿苷二磷酸半乳糖为添加剂冷藏兔血小板的研究

本研究探讨以尿苷二磷酸半乳糖(UDP-Gal)为添加剂的血小板冷藏保存方法。采集兔心脏血,按常规方法制备浓缩血小板悬液(PC),在悬液中添加UDP-Gal,放4℃冰箱储存10天。尔后观察血小板(Plt)数量?血小板平均体积(MPV)、血小板体积分布宽度(PDW)?血小板聚集功能(PagT)?血小板促凝活性实验(PF3aT和APCT)和血小板凋亡(apoptosis)的变化。将冷藏兔血小板用Cr51标记后,检测输入兔体内后的血小板生存时间。结果表明加入UDP-Gal冷藏保存10天后的PC的Plt、MPV、PDW、血小板促凝血活性与新鲜PC相比均无显著性变化(P(0.05);而冷藏对照PC的Plt明显减少,MPV、PDW显著增大,血小板促凝血活性减低,与新鲜PC相比差异有显著性(P>0.01);加入UDP-Gal的PC冷藏保存10天后的血小板凋亡率与新鲜PC相比有所增高(P(0.05),但明显低于冷藏对照组(P(0.01),其血小板聚集率(诱聚剂为阳离子没食子酸丙酯,C-PG)减低,但仍能保持在新鲜血小板的50%以上。添加UDP-Gal时冷藏保存10天的血小板与新鲜的血小板在体内的生存时间相近(P(0.05),输注兔体内72小时时的血小板存活率在新鲜对照组、UDP-Gal冷藏保存组和冷藏对照组分别为57.5%±7.2%、50.3%±6.3%和0.1%±0.5%。结论尿苷二磷酸半乳糖对冷藏保存的兔血小板有保护作用,并能延长冷藏兔血小板在体内的生存时间。     

Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: enhanced retention of in vitro functional activity

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long-term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second- messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single- donor platelet units (n = 8) were divided into three samples and treated with 6-percent dimethyl sulfoxide (DMSO), 2-percent DMSO or the platelet storage solution and 2-percent DMSO. The samples were placed directly into a -80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2-percent DMSO displayed statistically higher retention of functional activity and viability–including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response–than did platelets stored by the method using 6-percent DMSO. In addition, the treated platelets displayed statistically lower expression of p- selectin. The treated platelets showed no loss of cell number, > 88- percent retention of discoid morphology, and > 75-percent retention of ristocetin-induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.     

二甲亚砜与海藻糖联用对冷冻保存血小板的保护作用

本研究观察二甲亚砜（DMSO）与海藻糖联用对冷冻保存血小板的保护效果。实验分设空白组、海藻糖组、DMSO组、5％DMs0加海藻糖联用组、2．5％DMSO加海藻糖联用组。各组血小板置-80℃冰箱保存,37℃水浴融化。采用血细胞计数仪测定血小板回收率和MPV值、电子显微镜观察血小板超微结构变化和流式细胞仪测定血小板的CD41、CD42b、CD61及CD62p的表达水平。结果表明：海藻糖单独应用对提高回收率的保护作用不强,但海藻糖处理后冷冻保存的血小板的形态接近正常。DMSO在保证冷冻保存血小板的回收率和血小板整体完好性方面作用较为突出,但其血小板形态偏向于肿胀,仍有部分血小板呈异形性改变。DMSO和海藻糖合用对维持血小扳外部形态和内部结构接近正常稳态方面具有保护作用,同时保证冷冻保存血小板具有理想的回收率和较高的CD41、CD42b、CD61、CD62p表达水平。结论：DMSO和海藻糖联合应用对冷冻保存血小板具有协同保护作用,但DMSO和海藻糖单用或合用都较难抑制冷冻保存血小板的活化,两者合用作为血小板冷冻保护剂有望促使冷冻保存血小板临床输注效果的进一步提高。     

冷冻血小板的稳定性研究

目的探讨冷冻血小板融化复苏后的稳定性。方法将血小板分别置于-80℃和-85℃～-120℃保存，观察冷冻血小板融化复苏后纤维蛋白及絮状不可逆聚集的情况。结果-80℃保存血小板纤维蛋白及絮状不可逆聚集率为24．0％，～85℃～-120℃保存血小板纤维蛋白及絮状不可逆聚集率为0。血小板保存温度不同，纤维蛋白及絮状不可逆聚集发生率的差异有显著性（Х^2-243．18，P〈0．01），冷冻血小板冷冻速度和保存温度对血小板质量的影响有显著性。结论冷冻血小板于-85℃～-120℃保存的稳定性更好。     

利用SCID小鼠模型评价-80℃冷冻人血小板的体内生存能力

为了评估人血小板-80℃冷冻保存后在活体内的生存能力,取新鲜机采血小板加入二甲基亚砜（DMSO）,使其终浓度为5％,于～80℃保存10天后,取出立即放置于37℃水浴箱中快速解冻,并离心浓缩10倍。用带有超细针头的胰岛素注射器抽取浓缩血小板1130μl,经尾静脉注入重症联合免疫缺陷（SCID）小鼠体内,在注入0．5、2、4、6、12、24小时时采集小鼠全血,肝素抗凝,用CD61-PE标记后,流式细胞术计数人血小板,以30分钟时的人血小板计数为100％,计算人血小板存活率。结果表明：冷冻血小板在活体内存活率明显降低,新鲜血小板和冷冻血小板输注SCID小鼠体内4小时时的存活率分别为（79．5±9．1）％和（40．6±6．6）％（P〈0．01）,推算半寿期（T1/2）分别为7小时和2．5小时。结论：血小板-80℃冷冻保存后在活体内的生存能力降低。     

血小板保存3天后再冷冻保存的实验研究及临床应用

本研究探讨血小板常温保存3天后再进行-80℃冰箱内冷冻保存及临床应用的可行性。对当天冷冻和保存3天后再冷冻血小板进行了计数，并检测聚集力、黏附力以及CD62p的表达，并通过可对比性病例观察保存3天后再冷冻与当天冷冻血小板，临床应用的可能性。结果表明：在保存期3天之内血小板数量、低渗性休克反应、黏附功能无显著差异性改变（P〉0．05），但聚集功能和CD62p的表达率的变化有显著性差异（P〈0．05）。当天保存并冷冻的血小板与保存3天后再冷冻的血小板各项指标的变化都无显著性差异。CD62p的再表达率（CD62p凝血酶激活后阳性率-CD62p激活前的阳性率）也无显著性差异，分别是51．1±4．5和51．1±4．4（P〉0．05）。临床应用当天冷冻和保存3天后再冷冻血小板的CCI值分别是48％和49％，无显著性差异（P〉0．05）。结论：血小板保存3天后可以再-80℃冷冻保存，其临床应用效果与当天冷冻血小板比较后无明显差异。     

Correlation of in vivo and in vitro functions of fresh and stored human platelets

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.     

Effects of the addition of second-messenger effectors to platelet concentrates separated from whole-blood donations and stored at 4 degrees C or -80 degrees C

BACKGROUND: Platelet concentrates (PCs) are currently stored at 22 degrees C under continuous agitation. Because of the potential risk of the overgrowth of bacteria in case of contamination, PC shelf life is limited to 5 days. A mixture of second-messenger effectors is being evaluated to determine if it has benefits for cold liquid storage and cryopreservation of platelets. STUDY DESIGN AND METHODS: PCs separated from whole-blood donations by the buffy coat method were randomly assigned (n = 6 each) to be stored for 5 days at 22 degrees C under continuous agitation or at 4 degrees C after treatment with a platelet storage medium (ThromboSol, LifeCell Corp. ). PCs were also cryopreserved with 6-percent DMSO (final concentration) or with ThromboSol plus 2-percent DMSO (final concentration) (TC). After storage, platelets were analyzed by flow cytometry, transmission electron microscopy, and aggregation and perfusion techniques. RESULTS: Cold liquid storage of ThromboSol-treated platelets resulted in a lower binding of coagulation factor Va on the platelet surface than on platelets stored at 22 degrees C. In transmission electron microscopy, a conversion to spherical morphology was seen in the case of cold liquid storage. No difference between ThromboSol-treated platelets stored at 4 degrees C and platelets stored at 22 degrees C was seen in perfusion studies. Cryopreservation in the presence of TC prevented the reduction in glycoprotein Ib and IV expression on platelet surface that is seen in 6-percent DMSO-cryopreserved platelets. Platelets cryopreserved in TC covered, by thrombus, a significantly greater percentage of the perfused surface after the freezing and thawing process. CONCLUSION: ThromboSol-treated PCs separated from whole-blood donations by the buffy coat method, stored at 4 degrees C for 5 days, or cryopreserved in the presence of TC, maintained in vitro functional activity comparable to that achieved by current methods of storage, although discoid morphology was not preserved during cold liquid storage with ThromboSol.     

Heterogeneity of antibody response to human platelet transfusion.

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.     

低温保存血小板的制备与质控效果的观察

本研究探讨建立系统的二甲亚砜 (DMSO)低温保存血小板的制备及质控的技术和方法 ,以保证低温保存血小板的质量。采取的方法 :①DMSO在使用前进行超滤替代消毒 ;②采血后 6小时内对血液进行离心 ,将血袋管道系于套桶上方 ;③以 4 80×g的相对离心力 ,离心加速 9档 ,减速 4档 ;④分离血小板时流速不能太快 ,80 - 10 0ml富含血小板血浆应在 1分钟左右分完 ;在加入DMSO时速度以 1毫升 /分钟为宜 ;加好DMSO后放入 - 80℃冰箱保存 ;临床输注前将血小板置于 38- 4 0℃的循环水浴中 ;⑤融化后进行血小板计数、白细胞和红细胞计数 ,检测HBsAg、抗 HCV、抗 HIV、梅毒特异性抗体 ,测定丙氨酸氨基转移酶 (ALT)并进行细菌培养。结果表明 :共制备14 80 0单位低温保存血小板 ,其中机采血小板 80单位。对其中 30 0单位进行质控。手工分离血小板计数≥ 2 .4×10 10 /单位 ,平均得率在 70 %以上。机采血小板计数≥ 2 .5× 10 11/单位。手工分离血小板红细胞污染数≤ 1× 10 9个/单位 ,白细胞污染数≤ 5× 10 7个 /单位。融化后进行细菌培养 ,无细菌生长 ;ALT均在正常范围内。患者输注后具有明显的止血作用。结论 :本方法制备的手工分离和机采低温保存血小板质量可靠 ,临床应用效果良好。